The Adaptor Protein Grb2b is an Essential Modulator for Lympho-venous Sprout Formation in the Zebrafish Trunk

Vegfc/Vegfr3 signaling is critical for lymphangiogenesis, the sprouting of lymphatic vessels. In zebrafish, cells sprouting from the posterior cardinal vein can either form lymphatic precursor cells or contribute to intersegmental vein formation. Both, the Vegfc-dependent differential induction of Prox1a in sprouting cells as well as a Notch-mediated pre-pattern within intersegmental vessels have been associated with the regulation of secondary sprout behavior. However, how exactly a differential lymphatic versus venous sprout cell behavior is achieved is not fully understood. Here, we characterize a zebrafish mutant in the adaptor protein Grb2b, and demonstrate through genetic interaction studies that Grb2b acts within the Vegfr3 pathway. Mutant embryos exhibit phenotypes that are consistent with reduced Vegfr3 signaling outputs prior to the sprouting of endothelial cells from the vein. During secondary sprouting stages, loss of grb2b leads to defective cell behaviors resulting in a loss of parachordal lymphangioblasts, while only partially affecting the number of intersegmental veins. A second GRB2 zebrafish ortholog, grb2a, contributes to the development of lymphatic structures in the meninges and in the head, but not in the trunk. Our results illustrate an essential role of Grb2b in vivo for cell migration to the horizontal myoseptum and for the correct formation of the lymphatic vasculature, while being less critically required in intersegmental vein formation. Thus, there appear to be higher requirements for Grb2b and therefore Vegfr3 downstream signaling levels in lymphatic versus vein precursor-generating sprouts.